Cell surface glycan engineering reveals that matriglycan alone can recapitulate dystroglycan binding and function

α-Dystroglycan (α-DG) is uniquely modified on O-mannose sites by a repeating disaccharide (-Xylα1,3-GlcAβ1,3-)n termed matriglycan, which is a receptor for laminin-G domain-containing proteins and employed by old-world arenaviruses for infection. Using chemoenzymatically synthesized matriglycans printed as a microarray, we demonstrate length-dependent binding to Laminin, Lassa virus GP1, and the clinically-important antibody IIH6. Utilizing an enzymatic engineering approach, an N-linked glycoprotein was converted into a IIH6-positive Laminin-binding glycoprotein. Engineering of the surface of cells deficient for either α-DG or O-mannosylation with matriglycans of sufficient length recovers infection with a Lassa-pseudovirus. Finally, free matriglycan in a dose and length dependent manner inhibits viral infection of wildtype cells. These results indicate that matriglycan alone is necessary and sufficient for IIH6 staining, Laminin and LASV GP1 binding, and Lassa-pseudovirus infection and support a model in which it is a tunable receptor for which increasing chain length enhances ligand-binding capacity.


Chemical Synthesis
General methods and materials 1 H and 13 C (data from HSQC) NMR spectra were recorded on a Varian INOVA 300 MHz ( 13 C, 75 MHz), Varian INOVA 500 MHz, a Varian INOVA 600 MHz or an Agilent 900 MHz DD2 spectrometer with a triple resonance (HCN) cryogenically cooled probe spectrometer. Chemical shifts are reported in parts per million (ppm) relative to residual solvent signals used as the internal standard. NMR data is presented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, dd = doublet of doublet, m = multiplet and/or multiple resonances), integration, coupling constant in Hertz (Hz). All NMR signals were assigned on the basis of 1 H NMR, COSY, zTOCSY, gHSQCAD and gHMBCAD experiments. Mass spectra were recorded on a Shimadzu LCMS-IT-TOF mass spectrometer or an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). Reagents were purchased from Sigma-Aldrich (unless otherwise noted) and used without further purification. CH2Cl2 was freshly distilled from calcium hydride under nitrogen prior to use. Molecular sieves (4Å) were flame activated under vacuum prior to use. All moisture sensitive reactions were carried out under an argon atmosphere. HILIC-HPLC purification of compounds was performed on a Shimadzu 20AD UFLC LCMS-IT-TOF with a Waters XBridge BEH, Amide column, 5 μm, 10 x 250 mm or a SeQuant® ZIC®-HILIC column, 5 μm, 10 x 250 mm. HPLC grade acetonitrile and water were purchased from Fischer. β-Galactoside α-2,6sialyltransferase 1 (ST6GAL1) was generously provided by Dr. Kelley W. Moremen (Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA). Calf intestinal alkaline phosphatase (CIAP) was purchased from sigma. Clostridium perfringens (C. perfringens) neuraminidase was purchased from New England BioLabs. UDP-Glucuronic Acid was purchased from Sigma. UDP-Xylose was purchased from Carbosource (University of Georgia). S8 5'-Azidopentyl-β-D-xylopyranoside (12) 1,2,3,4-tetra-O-acetyl-β-D-xylopyranoside 11 (1.5 g, 4.7 mmol) and 5-azidopentanol (913 mg, 7.1 mmol) were dissolved in anhydrous CH2Cl2 (20 mL) with 4Å molecular sieves and stirred under argon for 30 mins. The mixture was cooled to 0°C and boron trifluoride diethyletherate (1.74 mL, 14.1 mmol) was added dropwise over 15 min and the reaction was stirred while slowly warming to room temperature overnight. The reaction mixture was then diluted with CH2Cl2, filtered through Celite, washed with saturated NaHCO3, brine, then dried with MgSO4, filtered, and then concentrated in vacuo. The crude product was dissolved in a solution of sodium methoxide in methanol (5 mL) and stirred for one hour at room temperature. The solution was then neutralized with Amberlite® IR-120 (H + ) ion-exchange resin, filtered and concentrated. Purification by silica gel column chromatography (

Enzymatic Synthesis
General procedure for the installation of β1,4-GlcA using B4GAT1 Xylose acceptor (10.6 μmol) and UDP-GlcA (15.9 μmmol) were dissolved at a final xylose-derivative concentration of 10 mM in a MOPS buffered solution (100 mM, pH 7.0) containing MnCl2 (10 mM). CIAP (1% total volume) and B4GAT1 (43 μg/μmol acceptor) were added, and the reaction mixture was incubated overnight at 37°C with gentle shaking. Reaction progress was monitored by ESI-MS and if starting material remained after 18 h another portion of B4GAT1 was added until no starting material could be detected. The reaction mixture was centrifuged using a Nanosep® Omega ultrafiltration device (10 kDa MWCO) to remove enzymes and the filtrate was lyophilized. The residue was purified by HPLC using a SeQuant ZIC-HILIC Amide column (5 μm, 10 ✕ 250 mm) with 1% of the flow diverted to the ESI-MS detector. Mobile phase A was ammonium formate in water (10 mM, adjusted to pH 4.5 with formic acid); Mobile phase B was a mixture of acetonitrile (90%) with ammonium formate in water (10%, 10 mM, pH = 4.5 with formic acid). The following gradient was used to provide the desired product: 1) Gradient of 90% to 60% mobile phase A from 0 -35 min; 2) gradient of 60% to 30% mobile phase A from 35 -40 min; 3) 30% mobile phase A from 35 -55 min; 4) gradient of 30% to 90% mobile phase A from 55 -60 min.
The residue for reactions yielding matriglycans 8 was purified by HPLC using a SeQuant ZIC-HILIC Amide column (5 μm, 10 ✕ 250 mm). The residue for reactions yielding matriglycans 3 was purified by HPLC using Waters XBridge BEH, Amide column (5 μm, 10 ✕ 250 mm). 1% of the flow diverted to the ESI-MS detector. For all HPLC purifications, mobile phase A was ammonium formate in water (10 mM, adjusted to pH 4.5 with formic acid); Mobile phase B was a mixture of acetonitrile (90%) with ammonium formate in water (10%, 10 mM, pH = 4.5 with formic acid). The following gradient was used for both columns to provide the desired products: 1) Gradient of 90% to 60% mobile phase A from 0 -35 min; 2) gradient of 60% to 30% mobile phase A from 35 -40 min; 3) 30% mobile phase A from 35 -55 min; 4) gradient of 30% to 90% mobile phase A from 55 -60 min. Fractions were collected with a volume of approximately 250 μL (20 sec intervals) and products were confirmed by ESI-MS before pooling and lyophilizing. HPLC-MS analysis and observed MS values for matriglycans are shown in Supplementary  Fig. 1  General protocol for conjugation of matriglycans to CMP-Neu5Az by CuAAC Stock solutions of 0.1 M CuSO4, 0.2 M sodium L-ascorbate and 0.1 M TBTA in 0.1 M NH4HCO3 were freshly made before each CuAAC reaction. 2 equivalents of CuSO4 per GlcA-carboxylate residue were used for each reaction. Sodium ascorbate and TBTA were adjusted to CuSO4 quantities at a ratio of 1.5:1 for sodium ascorbate/CuSO4 and 0.5:1 for TBTA/CuSO4. CuSO4, sodium ascorbate and TBTA were pre-mixed by vortexing, and were then added to a solution of alkyne-matriglycans 8a-i (1 equivalent) and CMP-Neu5Az 9 2 (3 equivalents) in 100 µL 0.1 M NH4HCO3. The resulting mixture was stirred at room temperature for 2 hours to have minimal hydrolysis of the CMP-Neu5Ac-derivative. The mixture was then directly loaded onto a P2-BioGel column kept at 4°C and the product was purified using 0.1 NH4HCO3 as eluent, analyzed by ESI-MS and immediately lyophilized and used for glycoengineering studies.

Transfer of Matriglycan Oligosaccharides to N-linked Glycopeptide 11
Disaccharide acceptor 7 (0.2 μmol) was dissolved at a concentration of 10 mM in a MES buffered solution (100 mM, pH 6.0) containing MnCl2 (10 mM), UDP-Xyl (1.2 μmol) and UDP-GlcA (1.4 μmol). CIAP (1% total volume) and LARGE1 (100 μg/μmol acceptor) were added, and the reaction mixture was incubated overnight at 37 °C with gentle shaking. The reaction mixture was centrifuged using a Nanosep® Omega ultrafiltration device (30 kDa MWCO) to remove the enzymes, and the resulting filtrate was lyophilized. The residue was purified by P-2 Bio-Gel column chromatography using 0.1 NH4HCO3 as eluent. Matriglycan containing fractions were combined and lyophilized to give a mixture of 8a-e (0.6 mg), which was used without further purification. The mixture of matriglycans 8a-e after P-2 Bio-Gel purification was analyzed by LC-MS using a SeQuant ZIC-HILIC Amide column (3.5 μm, 2.1 ✕ 150 mm). Mobile phase A was ammonium formate in water (10 mM, adjusted to pH 3.4 with formic acid); Mobile phase B was 100% acetonitrile. The following gradient was used for the analysis: 1) Gradient of 85% mobile phase B from 0 -5 min; 2) gradient of 85% to 30% mobile phase B from 5 -40 min; 3) 30% mobile phase B from 40 -50 min; 4) gradient of 30% to 85% mobile phase B from 50 -55 min. 5) gradient of 85% mobile phase B from 55 -60 min. HPLC-MS analysis is shown in Supplementary  Fig. 5.
The conjugation of matriglycans (8a-e mixture) to CMP-Neu5Az 9 was performed according to the protocol for click-conjugation by CuAAC to yield 10a-e (0.4 mg). ESI-MS analysis of the 10a-e mixture is shown in Supplementary Fig. 6.